Serotonin (5-hydroxytryptamine, 5-HT) is a critical monoamine neurotransmitter in the central nervous system that plays a key role in regulating emotion, mood, and sense of wellbeing. Serotonergic neurons synthesize, store, transport, and release serotonin analogous to other specialized neurons that govern only a single, unique type of monoamine neurotransmitter. The biosynthesis of serotonin transpires within the cell bodies of serotonergic neurons, which are localized to discrete cell clusters within the brainstem. The vesicular monoamine transporter (VMAT) protein stores serotonin within secretory vesicles at high concentrations (50-270 mM) and low pH (˜5). Secretory vesicles transport serotonin to axon terminals that innervate most brain regions, thereby maintaining the vesicle pool size and its stores at steady-state levels for continued release of vesicular serotonin into the synaptic cleft.
Deficient serotonin levels are implicated in the etiology of an array of debilitating neuropsychiatric disorders which include anxiety, bipolar disorder, and clinical depression. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed class of psychotropic medications and utilized as first-line agents to elevate serotonin levels. The persistent administration of SSRIs to serotonergic neurons indirectly dampens negative feedback sensitivity to serotonin release, thereby upregulating the synthesis and transport of serotonin. Accordingly, the enhanced activities promote higher vesicle stores and mobilize a larger vesicle pool size in order to accommodate the progressive release of vesicular serotonin.
Conventional molecular imaging tools for monitoring vesicular serotonin levels or the efficacy of SSRIs to modulate the vesicle pool size and its stores primarily include serotonin autofluorescence, fluorescent probes, and radiolabeled ligands. Unfortunately, the conventional technologies present certain drawbacks such as displaying limited selectivity, allowing for only indirect observation of serotonin, or requiring the use of invasive biomedical devices.
Recent developments in fluorescent molecular sensors have allowed for the selective labeling and direct visualization of similar monoamine neurotransmitters in neuroimaging applications. For such applications, fluorescent molecular sensors that can absorb and emit light within the near-infrared (NIR) optical imaging window (600-900 nm) are highly desirable because NIR light affords limited background fluorescence and high penetration depths in biological samples upon irradiation.
A water-soluble fluorescence-based molecular sensor would permit a unified approach in the selective recognition and visualization of serotonin in vivo and in vitro analyses that limits interference with native neuronal functions. Moreover, a fluorescence-based turn-on molecular sensor possessing spectroscopic properties capable of fluorescence emission in the near infrared (NIR) spectral region (i.e., a wavelength emission greater than 600 nm) would be particularly advantageous by minimizing background from biological analytes (e.g., riboflavin), reducing photodamage to biological samples, and allowing for greater tissue penetration.
Although fluorescence-based technologies remain a compelling approach to selectively detect and image serotonin, they have not been a practical approach toward in vivo and ex vivo analyses because they either resulted in biosensors that could not be used in biological models or did not provide a fluorescence turn-on response due to the photophysical tendency of the electron-rich indoleamine moiety to quench the fluorescence response of the fluorophore through photoinduced electron transfer (PET). For example, the NeuroSensor 521 (NS521)-based platform (a coumarin-3-aldehyde scaffold having the following structure) was developed to provide a variety of fluorescence-based turn-on molecular sensors for selective recognition of the catecholamine-based neurotransmitters, norepinephrine and dopamine, and integrated into a tailored approach to comprise a method for the selective labeling and imaging of norepinephrine in secretory vesicles of live and fixed cells that allows for discrimination between secretory cell populations (phenotypes) and continuous observation of neurotransmitter trafficking. See PCT/US2014/031490 filed Mar. 21, 2014, which is incorporated by reference in its entirety. For reference, the structure of NS521 is shown in FIG. 1 (A). In particular, it was discovered that the selection of an appropriate pendant aryl substituent (e.g., p-methoxyphenyl) allows NS521-based compounds to display a marked turn-on fluorescence response separately toward both catecholamines, which typically quench fluorescence emission. Unfortunately, serotonin strongly quenched the fluorescence response of all the compounds based on the NS521 platform.
Therefore, a need exists for a molecular sensor that demonstrates a turn-on NIR fluorescence response upon selective detection of biological amines, such as serotonin.